Invasive chemometry

ABSTRACT

The invention relates to methods and devices for assessing one or more components of a selected tissue in an animal. The present invention permits non-invasive assessment of tissue components in a body structure containing multiple tissue types by assessing multiple regions of the animal&#39;s body for an optical characteristic of the tissue of interest and separately assessing one or more optical (e.g., Raman or NIR) characteristics of the tissue component for one or more regions that exhibit the optical characteristic of the tissue of interest.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending U.S.application Ser. No. 11/146,458, which was filed on 7 Jun. 2005, theentire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The invention relates generally to the field of in vivo chemometricanalyses of chemical components of cells, tissues, or organs in a livingorganism.

Analyses of the chemical composition of blood and other tissues areamong the most commonly performed medical diagnostic techniques.Typically, such analyses are performed by obtaining a sample of thetissue to be analyzed from a patient (e.g., by drawing blood or byperforming a biopsy) and thereafter subjecting the sample to variousanalytical techniques. Such invasive techniques have disadvantagesincluding discomfort to the patient during sample collection,inconvenience of sample collection, and the possibility that collectedsamples can be lost or misidentified. Discomfort and inconvenience aremagnified in situations in which frequent or regular sample collectionis required, such as with blood glucose determinations for diabeticpatients.

Various analytical chemical techniques are known for quantitation ofindividual chemical species, but most such techniques quantify only oneor a few chemical species independently or one at a time. Among otheranalytical techniques that are known are a variety of spectraltechniques, including those involving absorbance, transmittance,reflectance, emission, and scattering (elastic and non-elastic) ofradiation applied to a sample. For example, Raman scattering analysis ofwhole blood has been described (Enejder et al., 2002, Optics Lett.27(22):2004-2006) and is suitable for clinical quantitation of bloodglucose, dissolved oxygen, dissolved carbon dioxide, urea, lactic acid,creatine, bicarbonate, electrolytes, protein, albumin, cholesterol,triglycerides, hematocrit, and hemoglobin. Spectral techniques, such asRaman spectral analysis, have the advantage that multiple chemicalspecies can be quantified simultaneously, so long as the species can bespectrally distinguished.

Others have described analytical devices and techniques intended fornon-invasive in vivo analysis of tissue components. However, each ofthese has certain disadvantages and limitations. For example, each ofBerger et al. (U.S. Pat. No. 5,615,673) and Yang et al. (U.S. Pat. No.6,167,290) describes a Raman spectroscopic system designed fortransdermal analysis of blood components. Xie (U.S. Patent ApplicationPublication No. 2005/0043597) describes a spectral analysis systemintended to analyze blood components using radiation passed across anail of a finger or toe. In each of these instances, individualvariation in skin or nail properties and in blood vessel placement cansignificantly affect the utility of the devices and methods.

A need exists for systems and methods for non-invasive compositionalanalysis of human tissues, particularly including blood. The presentinvention satisfies this need.

BRIEF SUMMARY OF THE INVENTION

The invention relates to method of assessing one or more components ofan animal tissue (e.g., a human tissue), either in vitro or in vivo. Themethod includes assessing multiple irradiated regions of an animal bodyto identify at least one region that exhibits a first opticalcharacteristic of the tissue. A second optical characteristic of thecomponent (e.g., a Raman shift characteristic of the component) isassessed for at least one identified region (and preferably for multipleregions identified as exhibiting the first optical characteristic). Inthis method, interfering signals from the same or a different componentin a tissue other than the tissue of interest can be reduced or avoided.In some embodiments, one or more confirmatory optical characteristics ofthe tissue can be assessed at the identified regions to confirm that theindividual identified regions correspond to the tissue of interest. Thesecond optical characteristic of the component can, if desired, beassessed only at identified regions that exhibit the confirmatoryoptical characteristic.

A variety of devices can be used to perform the assessment methodsdescribed herein. By way of example, the methods can be performed usinga device wherein the first optical characteristics of the irradiatedregions are assessed using multiple optical conduits (e.g., plastic-cladoptical glass fibers). These conduits optically couple the irradiatedregions with a detector. If desired, the conduits can optically coupleindividual irradiated regions with different detector elements of thedetector. If an image of the assessed regions of the animal body isdesired, then the arrangement of the optical conduits can be controlled,using a coherent bundle of optical fibers, for example.

If the assessed body regions are accessible from a surface of theanimal, then the conduits can simply be pressed against, or maintainedin close opposition to, the surface. Alternatively, the conduits canextend along a probe inserted into the body of the animal or appliedagainst a body surface of the animal. Any of a wide variety of knownprobe-type devices can be used, including probes in which opticalconduits are fixedly attached to the probe body and those in which theoptical conduits (or a sub-assembly including the optical conduits) canbe moved within the probe body such that the detection end of eachoptical conduit can be used to assess multiple body regions withoutmoving the probe body. By way of example, the optical conduits canfixedly or movably disposed within a catheter, a cannula, or anendoscope (e.g., an arthroscope, a bronchoscope, a thoracoscope, acolonoscope, a sigmoidoscope, a duodenoscope, a gastroscope, apancreatoscope, a choledochoscope, a nasopharyngoscope, arhinolaryngoscope, a laparoscope, or a colposcope).

In one embodiment, the optical conduits are rigidly fixed relative toone another (e.g., the optical conduits are fused or enclosed within anoptically clear jacket). Such fixation of conduits to one another canimprove the rigidity and durability of the conduits and can enhance thecoherence of the image produced using the conduits, in coherent imagingsystems. Significantly, the collection axes of the optical conduits neednot necessarily be substantially parallel, as in an imaging device. Atleast in embodiments in which a coherent image of the assessed bodyregions is not required, there need not be any particular relationshipamong the collection axes of the conduits. The axes can be substantiallyparallel, in dispersed parallel bundles, or irregularly arranged.Because the first and second optical characteristics are collected usingthe same optical fiber for any particular body location, there need beno particular geometric arrangement among the conduits.

The method described herein can be used to assess a wide variety ofcomponents in animal tissues, either individually or substantiallysimultaneously. The components can be individual chemical species (e.g.,one or more of glucose, dissolved oxygen, dissolved carbon dioxide,urea, lactic acid, creatine, bicarbonate, an electrolyte, protein,cholesterol, triglycerides, lipids other than triglycerides, andhemoglobin). The methods can also be used to assess cells in a tissue,and to distinguish normal and abnormal cells (or to detect the presenceof abnormal cells, such as cancer cells). Substantially any componenthaving one or more optical characteristics by which it may be identifiedcan be assessed in an animal tissue using the methods described herein.

Device useful for assessing a component of a tissue of an animal asdescribed herein should have a detector capable of identify regions ofan animal body that exhibits a first optical characteristic of a tissueof interest when the regions are irradiated. The device should also havea detector capable of assessing a second optical characteristic of thecomponent of interest in those body regions identified as correspondingto the tissue of interest. The same detector can be used for bothfunctions, or separate detectors can be used. The device should includea controller for limiting assessment of the second opticalcharacteristics to the identified regions. Preferably, the device alsoincludes optical conduits which transmit light reflected, transmitted,emitted, or scattered by or from the body regions to one or both of thedetectors. Although the device need not include a radiation source forgenerating the light to be reflected, transmitted, scattered, orabsorbed and re-emitted by the tissues and components, it is preferablethat a controlled (e.g., monochromatic) light source be used, and such alight source (e.g., a laser) can be incorporated into the device. In oneembodiment, optical illuminating fibers are used to transmit light fromthe light source to the tissue(s) and component(s) being assessed and aseparate set of optical detection fibers are used to transmit light fromthe tissue(s) and component(s) to the detector(s). One or more opticalfilters or other light-manipulating elements can be interposed among theother optical components. By way of example, an optical filter thatreduces or substantially prevents transmission of light having the samewavelength as that used to illuminate the sample can be used todistinguish Raman scattered light from illuminating light.

BRIEF SUMMARY OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram of an embodiment of the non-invasivedevice described herein.

FIG. 2 is an example of a layout pattern for optical fibers and holes ina device described herein.

FIG. 3 is an example of a layout pattern for optical fibers and holes ina device described herein.

FIG. 4 is an example of a layout pattern for optical fibers in a devicedescribed herein.

FIG. 5, comprising FIGS. 5A and 5B, is a diagram illustrating a detailof two embodiments of the device described herein.

FIG. 6 is a diagram illustrating a portion of an optical probe 20penetrating several tissues of an animal.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to methods and devices for assessing one or morecomponents of a tissue in an animal. Prior analytical methods requiredbiopsy and isolation of the desired tissue and subsequent analysis. Thepresent invention permits assessment of tissue components withoutremoval of the tissue from the animal body and without isolation of thetissue or components from other tissues or components with which it maybe interspersed.

Crudely simplified, the methods described herein involve assessing anoptical property (e.g., reflectance of a particular wavelength of light)for multiple regions of a portion of an animal's body, whether externalor internal. Regions of the body which exhibit an optical characteristicof one or more desired tissue types are selected for assessment of oneor more additional optical characteristics (e.g., Raman spectralcharacteristics). These additional characteristics can be selected toprovide information about the presence, concentration, or oxidationstate (for example) of one or more components of the animal's blood.Further by way of example, the methods and devices described herein canbe used by inserting a probe into an animal's body. Multiple opticalconduits on the probe can be used to irradiate multiple portions of thebody, and other optical conduits can be used to collect radiationreflected, transmitted, emitted, or scattered by irradiated portions thebody. Analysis of that collected radiation can indicate the type(s) oftissue from which the individual conduits are collecting radiation, andthose conduits can be used to collect radiation for which the opticalcharacteristics are informative of the presence, absence, state, orconcentration of a component of a tissue of interest.

These methods can be practiced using a device that optically analyzesmultiple regions of the body. The device is capable of detecting theoptical characteristic of tissues in an addressable manner, so that thedevice can distinguish regions based on the presence, absence,magnitude, or rate of change of the characteristic. The device canthereby identify regions of the body at which the tissue of interest ispresent. The device is also capable of detecting an opticalcharacteristic of a component of a tissue of interest for each of themultiple regions, thereby assessing the presence, absence, or relativeconcentration of the component for each region. By combining these twocapabilities, the device can assess the tissue component present at ornear regions of the body that include the tissue (and can disregardoptical characteristics of other components and tissues). As a result,noise, weak signals, and signals arising from compounds in tissues otherthan the tissue of interest can be avoided, and a signal correspondingto the desired component in the tissue of interest can be analyzed.

Definitions

As used herein, each of the following terms has the meaning associatedwith it in this section.

A “tissue” of an animal body means a collection of cells and/orextracellular materials that form a discernable body structure. In thissense, the word tissue is used in its usual sense in the medical arts,including structures composed almost entirely of living cells,structures composed almost entirely of non-living materials, andstructures composed of a mixture of living cells and non-livingmaterials. Non-limiting examples of tissues include epithelia, muscle,liver, blood, serum, bone, tendon, nerve, brain, and skin.

“Bandwidth” means the range of wavelengths in a beam of radiation,consistent with a specified full width at half maximum.

“Bandpass” of a detector or other system means the range of wavelengthsthat the detector or system can distinguish (i.e., transmit or permit topass through its optics), as assessed using the full width at halfmaximum intensity method.

The “full width at half maximum” (“FWHM”) method is a way ofcharacterizing radiation including a range of wavelengths by identifyingthe range of contiguous wavelengths that over which the magnitude of aproperty (e.g., intensity or detection capacity) is equal to at leasthalf the maximum magnitude of that property in the radiation at a singlewavelength.

An “optical characteristic” of a compound or tissue property is anoptical property of the compound or tissue by which the compound ortissue can be distinguished from other compounds or tissues that occurtogether with the compound or tissue of interest. By way of example, anoptical characteristic of blood is an optical property (e.g., absorbanceor reflectance in the red-to-near infrared (NIR) region of theelectromagnetic spectrum) that can be used to differentiate blood or ablood-rich tissue from tissues which contain little or no blood near avascularized surface of an animal. Similarly, an optical property of ablood component such as glucose is an optical property (e.g., a Raman orNIR spectrum) of a component of blood that can be used to differentiatethe component from other blood components.

A “region” in a sample refers to a relatively small area of anilluminated surface of an animal. For example, regions can have sizes of0.01-1 square millimeter. The geometry of the area corresponding to aregion is not critical. For example, a region can refer to a circular,annular, or square area of a surface. A region can be as small as thearea of a surface from which light is collected by a single opticalfiber or by a bundle of optical fibers (e.g., areas as small as a fewsquare microns).

The terms “optical” and “spectroscopic” are used interchangeably hereinto refer to properties of materials (and to methods of assessing suchproperties). The term “spectroscopic” is generally understood to referto the interaction of electromagnetic radiation, electrons, or neutronswith the materials. The term “optical” typically refers to aninteraction with electromagnetic radiation. For example, althoughelectron microscopy is not always commonly considered a “spectroscopic”or “optical” method, the two terms are used inclusively herein toencompass electron microscopy and other methods of assessing interactionof a material with visible, ultraviolet, or infrared light, withneutrons, or with other radiation.

The terms “light” and “radiation” are used interchangeably herein torefer to electromagnetic radiation having wavelengths associated withordinary spectrographic techniques, such as radiation in the ultraviolet(UV), visible, near infrared (NIR), and infrared (IR) regions of thespectrum. In particular, the term “light” is not limited to radiation inthe visible portion of the spectrum.

In the context of this application, an “optically clear” material is onewhich does not significantly inhibit transmission through the materialof radiation having a wavelength corresponding to an opticalcharacteristic of interest for a tissue of interest or for an analyzedcomponent of that tissue.

“Spectral resolution” means the ability of a radiation detection systemto resolve two spectral peaks.

“Quantification” of an optical characteristic of a compound meansassessment of the value of the characteristic with a greater precisionthan mere observation of the presence or absence of the compound.Quantification includes, for example, assessment of the characteristicwith sufficient precision that an approximate concentration of thecompound in a medium can be determined from a standard curve orassessment that the characteristic for one composition is greater orless than the characteristic for another composition.

Detailed Description

The invention relates to methods and apparatus for assessing a componentof one or more tissues of an animal. The methods involve assessing theoptical properties of irradiated portions of an animal body in order toidentify one or more regions of the body at which the optical propertiesof the tissue(s) of interest are evident. After those portions have beenidentified, an optical property of the compound of interest is assessedat some or all of those portions.

In one embodiment of the invention, multiple optical fibers arecontacted with (or brought into close opposition with) multiple portionsof a tissue that includes multiple cell types (e.g., the liver). Theportions are irradiated, and radiation collected from the optical fibersis analyzed to identify the portions which exhibit a first opticalcharacteristic of a desired cell type (e.g., hepatocytes). Radiationtransmitted from those identified portions by way of the optical fiberscan be analyzed for a second optical characteristic of a component ofinterest (e.g., glucose). By limiting assessment of the second opticalcharacteristic to optical fibers transmitting radiation from theidentified portions (i.e., from hepatocytes), glucose content inhepatocytes can be assessed, despite the fact that hepatocytes are notthe only cell type in liver tissue that contain glucose. Similarly,glucose content of red blood cells, leukocytes, Kupffer cells, bloodserum, or lymph in liver tissue can be assessed using the same opticalfiber probe—by analyzing the second optical characteristic (that ofglucose) using fibers which exhibit a first optical characteristic ofthe tissue for which glucose analysis is desired.

In another example of an embodiment of the invention, blood glucoseconcentration for a human can be measured by assessing visible lightscattered from multiple regions of a vascularized surface, such as skinor an oral inner cheek surface, to identify blood-rich portions (e.g.,portions of the surface at which a blood vessel lies very near thesurface). Raman-shifted radiation scattered from those portions can beassessed at Raman shift values characteristic of glucose and comparedwith reference values to estimate glucose content in the blood. Themethods and devices are not limited to detection of blood glucose.Substantially any optically-detectable component of blood can beassessed using the methods and apparatus described herein.

An important aspect of the invention is that a first optical property isassessed at multiple regions of the body to identify the location ofregions which exhibit one or more optical properties of a tissue ofinterest. By limiting assessment of an optical property of a tissuecomponent to these regions, signal strength can be improved and noiseand interference from cells, tissues, and compounds other than thecomponent of interest in the tissue of interest can be reduced.

Another important aspect of the invention is that the methods can beperformed either invasively or non-invasively. Substantially any animalsurface can be used, so long as it has the tissue of interestsufficiently close to the surface that one can assess optical propertiesof the tissue and component of interest using a non-invasive probe.Because optical conduits (e.g., plastic-clad optical glass fibers) canbe made exceedingly small, such conduits can be inserted into andthrough spaces, tissues, and fluids of animal bodies in a relativelyminimally invasive manner in order to assess portions of the body thatare not readily accessible using a probe applied to a body surface. Theavailability of highly sensitive detectors (e.g., charge-coupled device(CCD) detectors) and the ability of certain wavelengths of radiation topenetrate tissues without substantial absorption permit the methodsdescribed herein to assess tissue components in tissue located as farfrom a surface as the non-injurious intensity and absorbance of theradiation permits. By way of example, visible light can be used toassess tissue components tens or hundreds of micrometers from a surface,and infrared (e.g., including at least mid- and near-infrared light) canbe used to assess tissue components millimeters or centimeters distantfrom a surface.

The methods and apparatus described herein can be used to assesssubstantially any component of a tissue that can be spectrallydistinguished from other tissue components, either directly orindirectly. A tissue component can be assessed directly if it exhibitsat least one optical characteristic whereby it can be spectrallydistinguished from other tissue components. A tissue component can beindirectly assessed if an optical characteristic that can be spectrallydistinguished from optical characteristics of other tissue componentscan be associated with the component. By way of example, afluorescently-labeled antibody can be introduced into the tissue of apatient, whereupon the antibody binds with a protein which bears anepitope to which the antibody binds. Fluorescently-labeled proteins arethereby created, and those proteins can be detected using the methodsdescribed herein. Similarly, a compound that is selectively taken up bycells of a certain type can be used for indirect assessment of suchcells. Other indirect cell- and compound-labeling techniques are knownin the art, and substantially any of those techniques can be used inconjunction with the methods described herein.

Examples of tissue components that can be assessed using the methods anddevices described herein include whole cells (e.g., normal, cancerous,or other diseased cells), extracellular matrix materials (e.g.,collagens, atherosclerotic and other plaques, calcifications, bonematrix, and materials of exogenous origin such as plastic or metalfragments), and normal cellular components (e.g., glucose, dissolvedoxygen, dissolved carbon dioxide, urea, lactic acid, creatine,bicarbonate, electrolytes, proteins, nucleic acids, cholesterol,triglycerides, and hemoglobin).

The methods and devices described herein are essentially equallyapplicable to human and animal systems, the adaptations necessary forveterinary applications being readily evident to and capable of beingmade by an ordinary veterinarian.

Suitable Body Portions

The methods and devices described herein can be used to assess a tissuecomponent in substantially any body location, whether that location isreadily accessible from the outside of the animal or is deep within theinterior of the animal. Any body location which can be irradiated andfrom which transmitted, reflected, emitted, or scattered radiation canbe collected can be assessed using the methods and devices describedherein. In view of the information provided herein, choices ofanalytical devices and methods for performing the invention will beevident to a skilled artisan, the particular devices and methodsdepending on the identity of the tissue(s) and component(s) to beanalyzed. By way of example, liver tissue cannot ordinarily be accessedfrom the exterior of a healthy animal without making an incision,puncture, or other orifice by way of which an optical probe can becontacted with or brought into close opposition to liver tissue. Skin,for example, can normally be accessed externally without an incision orother surgically- or traumatically-created orifice.

It is not necessary that the body portion analyzed using the methods anddevices described herein be composed uniformly or entirely of a singlecell type. As described herein, multiple portions are assessed, andthose portions which do not exhibit an optical characteristic of thetissue of interest can be identified. Analysis of the component ofinterest can thereby be limited to a single tissue of interest. Becausemultiple body portions are analyzed, the methods described herein can beused to simultaneously assess one or more components in multiple tissuespresent at the analyzed body portions. By way of example, asubstantially cylindrical optical probe having multiple optical fiberbundles arranged therein such that the body portions from which thebundles collect radiation are circumferentially arranged (in an orderedor random manner) about the probe can be inserted into liver tissue.Some fibers or bundles will collect radiation only or substantially onlyfrom cells of a first type (e.g., hepatocytes). Other fibers or bundleswill collect radiation only or substantially only from tissue of asecond type (e.g., blood). Still other fibers or bundles will collectradiation only or substantially only from a fibrous or connectivetissue. The same component (e.g., glucose) or different components canbe assessed in these various tissue by performing the componentassessment using radiation collected by the corresponding fibers orbundles.

Vascularized Animal Surfaces

In one embodiment, the methods and devices described herein can be usedto assess a blood component in substantially any vascularized tissue. Itis recognized, however, that tissues that are not rich in blood canobscure or obstruct radiation transmitted to or from blood tissue. Forthat reason, the methods and devices described herein are preferablyused in connection with blood-rich tissues (e.g., arteries, veins,capillaries, and spaces in which blood can pool) and preferably avoid,to the extent practical, tissues that are not rich in blood. Tissuesthat contain significant amounts of connective tissue between ablood-rich tissue and the detector described herein are preferablyavoided.

Preferred tissue surfaces for assessment of blood components using themethods and devices described herein are those which are vascularizedand in which the vascularization is located relatively near the surfaceof the tissue. Although the methods described herein can be performedusing ordinary skin tissue (e.g., the tissue on the inner surface of theforearm or wrist), it is recognized that the keratinized surface ofskin, its connective-tissue rich dermal layer, and melanin and otherskin pigments can interfere with the methods. Non-dermal epithelia (e.g.an epithelium with a thin, or no, dermal layer underlying it) arepreferred surfaces for the assessment methods described herein.Likewise, epithelia that overlie vascularized tissue that is not coveredwith a keratinized layer of dead cells are preferable. Preferably, thetissue surface assessed does not have a keratinized layer of dead cells,an underlying dermal layer, or significant epithelial cell pigmentation.

Non-Invasive Chemometry

In one embodiment, the methods described herein are performednon-invasively. Substantially any surface of an animal can be analyzed,contingent on the presence of the tissue of interest at or near thesurface. For example, for blood analysis the apical surface (i.e., thefree surface; the surface opposite the basement membrane) of anon-dermal epithelium that is accessible without puncturing or cutting abody surface is preferred for such non-invasive methods. Numerous suchsurfaces are accessible on the human body. These surfaces are commonlythought of as “pink tissue” surfaces, and are generally moist,highly-vascularized tissues that line body orifices and cavities. Manyof these surfaces are mucosal epithelia, although vascularized surfaces(e.g., the superior surface of the tongue) that are not normallyconsidered mucosal epithelia are also suitable. Examples of suitablevascularized non-dermal epithelial surfaces include the floor of themouth, the soft palate, the lingual surface of the tongue, inner cheeksurfaces, the gums and gingiva, esophagus lining, stomach wall lining,intestinal and colonic linings, olfactory epithelium, pharyngealepithelium, bronchial epithelium, alveolar epithelium, urethralepithelium, vaginal epithelium, and vulval epithelium.

There are numerous advantages of performing the methods described hereinnon-invasively using a vascularized non-dermal epithelial surface. Inaddition to limiting spectral interference and improving thesignal-to-noise ratio for the desired analyte, analysis performed usinga non-dermal epithelial surface can be done relatively quickly andeasily and with a minimum of patient discomfort. The methods can beself-administered or administered to non-ambulatory patients.

Invasive Chemometry

The methods described herein can also be performed invasively, meaningthat at least one body structure of the animal is breached in order toplace the optical probe used in the methods described herein at aselected body location. The optical probe can be used to breach thetissue, in which instance, the probe should be constructed suitablyruggedly to withstand the forces of such breach without substantiallylosing or impairing its optical analytical functions. Alternatively, theoptical probe can be directed to a body location to which access hasbeen provided by breaching a body structure using an instrument otherthan the optical probe.

Use of an invasive probe has the advantage that multiple components ofmultiple tissues can be monitored sequentially or substantiallysimultaneously. The number of tissues that can be simultaneouslymonitored is limited by the number of tissues which can be put intocontact with or close opposition to one or more optical conduits of theprobe. FIG. 6 illustrates this concept. FIG. 6 is a simplified diagramshowing an optical probe 20 penetrating the skin S of an animal. Theprobe 20 also penetrates a connective tissue C and a muscle tissue M ofthe animal. Lenses 30 collect radiation from tissues which the probe 20contacts or is in close opposition to and transmit that radiation to oneor more optical conduits (not shown) within the probe 20. In FIG. 6,lens 30-1 collects radiation from skin tissue S that it contacts, lens30-2 collects radiation from connective tissue C that it contacts,lenses 30-3 and 30-4 collect radiation from muscle tissue M that theycontact, lens 30-5 collects radiation from radiation from venouscapillary B which lies in close opposition thereto, and lens 30-6collects radiation from arterial capillary A which lies in closeopposition thereto. As shown in FIG. 6, some lenses will collectradiation from more than one tissue type. For example, lens 30-7collects radiation from at least the muscle tissue M, arterial capillaryA (most likely including both arterial blood and capillary walltissues), and the fatty deposit F. The tissues corresponding to thevarious optical fibers can be identified and distinguished by analysisof the optical characteristics of radiation collected by the lenses 30and transmitted by way of the optical fibers.

One or more components of the devices described herein can be implantedand used in situ to monitor a tissue component, in conjunction withappropriate physical or transmitted (e.g., by radio waves) connectionsto the exterior of the patients body. Furthermore, the methods anddevices described herein can be used with an invasive probe, such as asheathed, drawn-optical fiber probe that pierces a tissue or is threadedalong a tubular body cavity such as a blood vessel. Theradiation-collecting optics corresponding to individual optical conduitsor bundles of optical conduits can be arranged on the probe in anorganized or random pattern. Other examples of devices suitable for usein the methods described herein include the microlens array fiber opticdevice described in co-pending U.S. patent application Ser. No.10/962,662, filed 13 Oct. 2004 and the chemical imaging fiberscopedescribed in U.S. Pat. No. 6,788,860.

Whether considered non-invasive or invasive, application of an opticalprobe as described herein to a body surface (e.g., the inner wall of thestomach by way of a gastroscope) is a convenient and minimally-traumaticway of performing the analysis described herein. Such analysis can beperformed by using an appropriate endoscope (e.g., one of anarthroscope, a bronchoscope, a thoracoscope, a colonoscope, asigmoidoscope, a duodenoscope, a gastroscope, a pancreatoscope, acholedochoscope, a nasopharyngoscope, a rhinolaryngoscope, alaparoscope, and a colposcope) to direct the optical probe to a bodylocation at or near which a tissue of interest occurs. If desired, animaging apparatus of the endoscope can be used to direct or confirmplacement of the optical probe. Alternatively, a cannula, catheter, orother device having a hollow through which the optical probe can bemoved can be used to place the optical probe at a desired body locationor to direct the optical probe to a desired site of tissue penetration.

Radiation Source

The body location at which a tissue component is to be assessed isirradiated. The irradiation can be applied to the same side of thetissue surface from which light is collected, from the opposite side(e.g., in the case of relatively thin accessible tissues such as theoral cheek), or some combination of these.

In most instances, light from a controlled source will be used toirradiate the surface. However, use of relatively uncontrolled radiationsources such as the sun or a household incandescent light bulb is notexcluded. In order to minimize variability, however, it is preferable touse light from a controlled source, such as a laser, light-emittingdiode, or a filament bulb. The controlled radiation source can beadapted to the geometry and sensitivity of the devices described hereinand can be selected based on the spectral properties of the tissuecomponent being analyzed. Certain spectroscopic techniques (e.g., Ramanscattering analysis) are best performed using substantiallymonochromatic light for irradiation of the sample. numerous suitablesources of substantially monochromatic light are known, including lasersand polychromatic light sources equipped with a diffraction grating, forexample. A skilled artisan is able to select one or more appropriateradiation sources based on the optical properties of blood, the opticalproperties of the tissue and component of interest, and thespectroscopic technique to be used to identify each.

The analytical methods described herein involve analysis of multipleregions of a sample. Those multiple regions are assessed for at leastthe occurrence of an optical characteristic of the tissue of interest,and some or all of the regions can be assessed for the occurrence of atleast one optical characteristic of the tissue component of interest.Preferably, a single light source is used for each of these analyses.However, multiple light sources can be used.

Each light source can be used to illuminate a portion of the samplesurface that includes all of the multiple regions. Alternatively, theradiation source can be used to illuminate only, or substantially only,the regions to be assessed. The assessed regions can be irradiatedsimultaneously, one at a time, in a random fashion, or otherwise. Forassessed regions that are determined not to exhibit an opticalcharacteristic of the tissue of interest, irradiation can bediscontinued, if desired, during analysis of regions at which the tissueof interest occurs. Alternatively, irradiation can be redirected fromregions that are determined not to exhibit an optical characteristic ofthe tissue of interest to regions that do, in order to boost theintensity of the optical signal from regions including the tissue ofinterest.

Unlike prior art methods, the methods described herein avoid much of theinterference and obstruction associated with keratinized and otherconnective tissue-rich portions of tissue surfaces. For this reason, itis not necessary, as it is with prior art methods, to select irradiationwavelengths that are not significantly absorbed by tissues other thanthe tissue of interest.

Irradiation wavelengths are not limited to the IR and NIR portions ofthe spectrum, and can include light of shorter wavelength, such as lighthaving wavelengths shorter than about 600 nanometers. Monochromaticlight having a wavelength in the range from about 600-800 nanometers issuitable for Raman spectral analysis of tissue, for example. Longerillumination wavelengths will, generally, induce less backgroundfluorescence in tissues (i.e., reducing the need to remove or correctfor fluorescently-emitted radiation emitted from the tissue), but candecrease the intensity of Raman scattered radiation. Selection of anappropriate illumination wavelength is within the level of ordinaryskill, taking into account the optical characteristics (e.g.,fluorescence, scattering, and absorption) of the tissue(s) and tissuecomponent(s) being assessed.

An advantage of using NIR radiation in the methods described herein isthat it penetrates biological tissues more deeply than visible light soas to enable assessment of tissue and components lying farther from thesample surface than is possible using shorter wavelengths. Appropriateselection of optical probe placement site can reduce the need fordeeply-penetrating irradiation. For example, for non-invasive analysisof a blood component, placement of the probe against a vascularized andpreferably non-dermal, non-keratinized epithelial surface alleviates theneed for NIR irradiation in many instances. In view of the increasednoise and interference that can be expected to result from analysis ofdeeply-penetrating radiation, it is preferable to use an optical probethat is placed as close as possible (preferably in contact with) thetissue of interest.

The radiation source can optionally be coupled with one or more lenses,beam splitters, diffraction gratings, polarization filters, bandpassfilters, or other optical elements selected for illuminating the samplesurface in a desired manner. Such optical elements and methods ofcoupling them with radiation sources are known in the art.

The devices described herein preferably include a radiation sourceselected for its suitability for analysis using the device.Alternatively, a device can be designed to analyze a sample usingordinary sunlight or other ambient light, such as residential lightingor an illuminating instrument ordinarily found in a doctor's office.

Optical Illumination Fibers

The surface or body location illuminated by the radiation source can bedirectly irradiated, that is by radiation transmitted through the airinterposed between the radiation source and the sample surface.Alternatively, radiation from the source can be transmitted to thesample surface by way of one or more optical fibers. The one or morefibers can be used to illuminate the surface continuously orintermittently over a portion that includes all of the regions assessedin the manner described below. Alternatively, one or more illuminatingfibers can be used to irradiate discrete (adjacent or non-adjacent)regions of the sample surface, and some or all of those irradiatedregions can be assessed in the manner described below. Devices andmethods for coupling optical fibers with radiation sources are known inthe art.

In one embodiment, one or more optical fibers used to illuminate thesample are bundled together with one or more optical detection fibersused to collect radiation reflected, emitted, or scattered from thetissue or its surface. Discrete bundles of illumination and detectionfibers can be directed to selected areas of the sample surface (e.g.,the bundles fixed in a selected geometric configuration and the ends ofthe bundles applied to or near the surface). The illumination fibers ineach bundle can transmit light to the corresponding selected area of thesurface, and light reflected, emitted, or scattered from that area ofthe surface can be collected by the detection fibers. Depending on thenature of the sample, light transmitted to the surface from theillumination fiber can pass through the surface and be reflected,scattered, or absorbed and emitted by one or more elements below thesurface within the sample. By way of example, light can penetrate thesurface of animal or plant tissues and reach cells or other structureswhich lie below the tissue surface. Interactions of subsurfacestructures with light transmitted through the surface can be assessedusing light transmitted back through the surface (or through a differentsurface of the sample) and collected by detection fibers. Lighttransmitted by the detection fibers of each bundle can be assessed in acombined or discrete fashion, as desired.

The optical illumination fibers can optionally be coupled with one ormore lenses, beam splitters, diffraction gratings, polarization filters,bandpass filters, or other optical elements selected for illuminatingthe sample surface in a desired manner. Such optical elements andmethods of coupling them with optical fibers are known in the art.

Detectors

Light is collected from the assessed regions of the vascularized surfaceof the animal and transmitted to one or more detectors. Preferably asingle detector is employed.

It is important that light be transmitted from the surface to thedetector in a “mappable” or “addressable” fashion, such that lighttransmitted from different assessed regions of the body can bedifferentiated by the detector. Differentiation of light from discreteassessed regions can be achieved by simultaneously transmitting lightfrom the regions to discrete portions (i.e., one or more detectionelements) of the detector. Such differentiation can also be achieved bytransmitting light from discrete regions to a single portion of thedetector, so long as the light from the discrete regions can bedifferentiated, such as by sampling different regions over time.Preferably, light from discrete assessed regions of a sample surface istransmitted separately to discrete portions of a detector having alinear or two-dimensional array of detector elements.

It is not necessary that the correspondence between a portion of adetector and the portion of the sample surface from which light istransmitted to that detector portion be known. The methods and devicesdescribed herein can be used so long as the correspondence between thedetector element(s) and a portion of the sample surface is the same forassessment of the optical characteristic of blood and the opticalcharacteristic(s) of a blood component. Likewise, there is norequirement that the relative two-dimensional locations of assessedregions on the sample surface be preserved on the corresponding portionsof, for example, a two-dimensional array of detector elements in adetector. If an image showing the optical characteristic of the tissueof interest or of a component of that tissue is desired to correspond tothe two-dimensional appearance of the surface, then the relativepositions of the assessed regions must be reflected in the relativepositions of the detector elements (if not in the same geometricpattern, then at least in a decodable pattern whereby the geometricarrangement of assessed regions can be reconstructed from the geometricarrangement of corresponding detector elements). By way of example, acoherent array of bundled optical fibers can be used to correlateassessed regions of a surface with corresponding regions of an image.

The detector(s) must be able to detect at least two types of opticalsignals. Preferably a detector capable of detecting both signals isused. First, the detector (hereafter referred to in the singular in thissection, regardless of whether one or more detectors is used) must beable to detect a first optical characteristic of the tissue of interest.Second, the detector must be able to detect a second opticalcharacteristic of the selected component of the tissue. In the methodsdescribed herein, the selected tissue component is assessed by detectingthe second characteristic only for assessed regions that exhibit thefirst characteristic. In this way, assessment of the selected tissuecomponent is performed only in tissues characterized by the presence ofdetectable tissue of interest.

Light detected by the detector can be light transmitted, reflected,emitted, or scattered by the tissue through air interposed between thetissue surface and the detector. Alternatively, the light can betransmitted by way of one or more optical fibers to the detector.Regardless of whether an optical fiber is employed, one or more otheroptical elements can be interposed between the surface and thedetector(s). If optical elements are used to facilitate transmissionfrom the surface to the detectors, any other optical element(s) can beoptically coupled with the fibers on either end or in the middle of suchfibers. Examples of suitable optical elements include one or morelenses, beam splitters, diffraction gratings, polarization filters,bandpass filters, or other optical elements selected for transmitting ormodifying light to be assessed by the detectors. Selection of one ormore appropriate optical elements and coupling of such elements with adetector and, optionally optical fibers, is within the ordinary level ofskill in this field.

By way of example, it is known that it is beneficial to use an opticalelement such as a filter, an interferometer or a dispersive spectrometerto detect Raman-shifted radiation scattered by a sample. For example, asuitable filter can be a cut-off filter, a Fabry Perot angle tunedfilter, an acousto-optic tunable filter, a liquid crystal tunablefilter, a Lyot filter, an Evans split element liquid crystal tunablefilter, a Solc liquid crystal tunable filter, or a liquid crystal FabryPerot tunable filter. Suitable interferometers include apolarization-independent imaging interferometer, a Michelsoninterferometer, a Sagnac interferometer, a Twynam-Green interferometer,a Mach-Zehnder interferometer, and a tunable Fabry Perot interferometer.

The construction and operation of the detector is not critical, so longas the detector is able to detect the relevant optical characteristic(s)described herein. Many suitable detectors are known in the art. It isalso known that detectors suitable for detecting certain relatively weakoptical emissions (e.g., Raman-shifted scattered radiation) can requirehighly sensitive detectors, such as charge-coupled device (CCD)detectors.

The detector is coupled with a controller of substantially any typesuitable for operation of the detector. The controller can be a programoperable on a personal computer, for example, or it can be a componentof a free-standing apparatus (e.g., a spectrometer) that includes thedetector. Optionally, the controller can operate other components of thedevice, such as a filter or a dispersive spectrometer.

In one embodiment, one or more optical fibers used to illuminate thesample are bundled together with one or more optical detection fibersused to collect radiation reflected, emitted, or scattered from thesurface. Discrete bundles of illumination and detection fibers can bedirected to selected areas of the sample surface (e.g., the bundlesfixed in a selected geometric configuration and the ends of the bundlesapplied to or near the surface). The illumination fibers in each bundletransmit light to the corresponding selected area of the surface, andlight reflected, emitted, or scattered from that area of the surface iscollected by the detection fibers. Light transmitted by the detectionfibers of each bundle assessed in a combined or discrete fashion, asdesired.

In another embodiment, assessed regions of the sample surface correspondto the areas from which individual optical detection fibers collectlight, and the light transmitted by each detection fiber is assessedseparately.

The assessed regions can together represent only a portion of the areaof the viewing field. It has been discovered that sampling the viewingfield at points representing a minority of the total area of the field(e.g., at two, four, ten, fifty, one hundred, or more regionsrepresenting, in sum, 25%, 5%, 1%, or less of the field) can yieldaccurate results. The shape of assessed regions is not critical. Forexample, circular, annular, oval, square, or rectangular regions can beassessed, as can the area (however shaped) from which light is collectedby a single detection fiber. Assessed regions can be adjacent oneanother, with no non-assessed region interposed between the adjacentassessed regions, whereby a substantially continuous patch or area of atissue surface can be assessed. Assessed regions which do not exhibit anoptical characteristic of the tissue of interest can be ignored forfurther analysis.

The area corresponding to each assessed region can be selected orgenerated in a variety of known ways. By way of example, a confocal maskor diffracting optical element placed in the illumination or collectionoptical path can limit illumination or collection to certain portions ofthe sample having a defined geometric relationship. Further by way ofexample, a plurality of regions can be assessed using a detectorcomprising a linear array of detector elements or a detector opticallycoupled with a linear array of optical fibers.

The number of regions of the sample surface that are assessed is notcritical. The maximum number of regions on a sample surface that can beassessed using a single multi-element detector will be determined by thenumber of detector units in the detector, the resolution and sensitivityof the detector and its associated optics, the sample size, the size ofthe assessed regions, the intensities and wavelengths of the light usedfor illumination and analysis, and other characteristics that areunderstood by the ordinary worker in this field. At least three regionsshould be assessed for occurrence of a first optical property—onecharacteristic of the tissue of interest (i.e., occurrence of that firstcharacteristic indicating that the tissue of interest is associated withthe region). Preferably, more (e.g., six, ten, twenty, or fifty or more)regions are assessed for occurrence of the first characteristic. Asecond optical property—one characteristic of the tissue component to beanalyzed—is assessed for at least one region at which the firstcharacteristic occurs. Confidence in the assessment of the component inthe tissue of interest can be increased by assessing the second propertyat multiple regions that exhibit the first characteristic.

Optical characteristics by which a tissue of interest can bedifferentiated from other tissues are known in the art. Selection of anappropriate characteristic for differentiating relatively tissues candepend on the type and nature of the tissue(s), and is within the levelof skill of the ordinary artisan in this field. Examples of opticalproperties of tissues that can be used to distinguish them includereflectance and Raman scattering characteristics. For example, forblood, these properties include:

i) the reflectance attributable to hemoglobin around a wavelength ofabout 700 nanometers (see, e.g., Solenenko et al., 2002, Phys. Med.Biol. 47:857-873);

ii) the Raman scattering peak near 1365 cm⁻¹ attributable to hemoglobin(this peak is nearer 1355 cm⁻¹ for deoxygenated hemoglobin and is nearer1380 cm⁻¹ for oxygenated hemoglobin)

Optical characteristics by which a component of a tissue can be assessedare known in the art. By way of example, Raman spectra of common bloodconstituents are disclosed in Enejder et al. (2002, Optics Lett.27(22):2004-2006), in U.S. Pat. No. 5,615,673, and in U.S. PatentApplication Publication no. 2005/0043597. NIR spectra of bloodcomponents are also reported in the literature. Use of this informationto identify and quantify components in a sample is within the level ofordinary skill in this field. Examples of blood components that can bedetected by Raman spectroscopy include glucose, creatine, lactic acid,carbon dioxide, and K, Mg, Na, Ca, and Cl ion complexes. Examples ofblood components that can be detected by NIR spectroscopy includeoxygenated and deoxygenated forms of hemoglobin. The methods describedherein are not limited to assessment of blood components, but can beused to assess any optically-assessable compound, structure, ormolecular state.

The methods and devices described herein can be used to detect normalcomponents of tissues and substances that do not naturally occur in thetissues of a healthy individual. By way of example, the presence,concentration, or both of a drug can be assessed in an individual'stissue. Further by way of example, metabolites associated withoccurrence of a disorder in a patient (e.g., acetone in muscle and bloodof patients afflicted with ketoacidosis) and pathogens (e.g., bacterialtoxins, bacterial cells, and viruses) can also be detected in anindividual's tissues. By assessing a Raman or NIR characteristic of redblood cells in an individual's blood, the individual's hematocrit can beassessed. Assessment of oxidized and reduced forms of electron chaincomponents can indicate the redox state of cells.

Light collected from multiple assessed regions of the sample can becombined prior to assessment of the optical property characteristic ofthe component. By way of example, light from all assessed regions thatexhibits an optical property characteristic of a tissue of interest canbe combined and the combined light can be assessed for the opticalproperty of a component of that tissue. Alternatively, assessed regionswhich exhibits an optical property characteristic of a tissue ofinterest can be identified, and the light from each of those regions canbe separately assessed for the optical property of the tissue component.

Multiple detectors can be used in the methods and devices describedherein (e.g., one for detecting the optical characteristic of the tissueof interest and another for detecting the optical characteristic(s) ofthe tissue component). If multiple detectors are used, then detectionelements of detectors for different optical properties should becorrelated such that detection elements receiving radiation from commonassessed regions can be identified. In that way, occurrence (ormagnitude or non-occurrence) of an optical property of the tissue ofinterest at an assessed region can be used to determine whether anoptical property of a tissue component from the same region should beassessed or recorded, for example. Use of a single, multi-purposedetector eliminates the need for such correlation. A detector havingdetection elements capable of detecting both radiation corresponding toan optical property of the tissue of interest and radiationcorresponding to one or more optical properties of a tissue componentthus eliminates the need for multiple detectors. A CCD detector capableof detecting both light reflected by the tissue of interest andRaman-shifted light scattered by a particular tissue component is anexample of a suitable detector.

Spectroscopic Analysis of a Blood Component

In one embodiment, the methods and devices described herein can be usedto identify portions of an animal tissue surface that contain (or arerelatively rich in) blood and to analyze one or more components of theblood in those portions. In many instances, blood can be distinguishedfrom other biological tissues relatively simply. By way of example,assessment of reflected radiation can be used to identify regions of atissue surface associated with blood (i.e., a surface overlying one ormore blood vessels or overlying a tissue containing pooled blood). Onceregions of a tissue surface associated with blood have been identified,further assessment of those regions can be performed to specificallyidentify, quantify, or both identify and quantify a component of bloodassociated with those regions.

The spectroscopic method used to assess the blood component is notcritical. Certain blood components (e.g., oxidized hemoglobin) havecharacteristic optical properties that can be assessed relativelysimply, such as by assuming that all of an optical property that isdetected is attributable to the component. However, assessment of manyindividual blood components (e.g., glucose, urea, or lactic acid) can besubject to significant interference from other compounds associated withthe region. In order to differentiate the blood component of interestfrom other compounds that may be present, use of spectroscopictechniques that are able to distinguish the component from othercompounds should be used.

Examples of highly specific spectroscopic techniques include Ramanspectroscopy and IR spectroscopy. NIR spectroscopy has sufficientspecificity for use in certain situations, such as differentiation ofoxygenated and deoxygenated hemoglobin. Each of these techniques isknown to be useful for correlating the presence of a specific compoundwith one or more detectable optical properties of that compound. Ramanspectroscopy often provides more information regarding the identity ofimaged materials than many other forms of spectroscopic analysis, soinclusion of Raman spectroscopy in the methods is preferred.

In an embodiment of the methods described herein, an opticallydetectable compound (e.g., a fluorescent dye or a compound with aneasily-detected Raman scattering characteristic) can be added to theblood of a subject prior to performing the methods described herein.Detection of the compound can indicate assessed regions of thevascularized surface overlying blood-containing vessels or tissues.Relatively blood-rich portions of the surface can be identified in thisway, and an optical characteristic of the blood component of interestcan be assessed from one or more of those portions. Examples of suitablefluorescent dyes that can be used in this manner include knownangiography dyes such as fluorescein and indocyanine green.

In addition to blood component-specific spectroscopic techniques, otherspectroscopic measurements (e.g., absorbance, fluorescence, and/orrefraction) can be performed to assess one or more of the regionssampled by the specific technique. This information can be used alone oras a supplement to the component-specific spectral information tofurther characterize the regions of the sample surface. This informationcan also be used to reduce the number of relatively blood-rich regionsat which the component-specific spectral analysis is performed,particularly if the concentration of the blood component is expected tobe non-homogenous in blood and one desires to assess suchnon-homogeneity.

Spectroscopic analysis of multiple regions of a tissue surface allowshigh quality spectral sensing and analysis without the need to performspectral imaging at every assessed region of a surface. The regionscorresponding to the presence of blood can be identified simply, andspectral analysis confined to those regions. Optical imaging can beperformed on the sample surface (e.g., simultaneously or separately) andthe optical image can be combined with selected blood component-specificspectrum information to define and locate regions of interest. Rapidlyobtaining spectra from sufficient different locations of this region ofinterest at one time allows highly efficient and accurate spectralanalysis and the identification of materials such as thrombi orpathogenic agents in blood.

Because a plurality of chemical compounds occur in blood and othertissues, it can be necessary to distinguish spectral features of theblood component of interest from overlapping spectral features of one ormore other compounds. Any of a variety of known methods can be used tocorrelate the spectrum obtained at any particular point with referencespectra collected or stored in a memory unit for the compounds. By wayof example, standard spectral library comparison methods can be used orthe spectral unmixing methods described in U.S. patent application Ser.No. 10/812,233, filed 29 Mar. 2004 can be used. Sampling multipleregions of a sample surface allows variations in the spectra collectedfrom the regions to be observed. Distinctions can be made as tocomponents present in the various regions of the sample. By way ofexample, it can be assumed that a component having spectral featuresthat do not vary in proportion to the relative amount of blood presentat the assessed region are not representative of the chemicalcomposition of blood, and can be considered background. For this region,it can be advantageous to assess optical properties of a tissue regionthat is determined to be relatively blood-poor or free of blood.

Correlative multivariate routines can be applied to spectral informationcollected from samples intentionally seeded with a known standardmaterial (e.g., a component deliberately added to blood in a knownamount). This approach incorporates calibration standards withinspectral information collected from a sample and permits quantitativechemical analysis.

Spectroscopic Analysis of a Component of a Tissue other than Blood

The methods and devices described herein can also be used to identifyportions of an animal tissue that contain (e.g., are, include, oroverlie) a tissue other than blood. Examples of such tissues includeepithelia, skeletal, smooth, and heart muscles, nerves, brain, bloodvessel walls, liver, pancreas, peritoneal membrane, ovary, connectivetissues (e.g., tendons and ligaments), and lymph nodes and vessels. Themethods and devices can also be used to detect and analyze the contentsof tissue-enclosed spaces, such as the interior of hair follicles, sweatglands, tissue inclusions, lipid bodies, pustules, blisters, subdermalnecrotic regions, mucoids secreted by goblet cells, the gall bladder,the pancreas, and the like. In each instance, a first opticalcharacteristic can be used to identify irradiated regions thatcorrespond to the tissue or space of interest. A second opticalcharacteristic of the identified regions (or of a component present atthose regions) can be assessed if desired.

The penetrating capacity of illuminating light depends on the intensityof the light, the wavelength of the illuminating radiation, and thetype(s) of tissue through which the light must penetrate, and theserelationships are known in the art. By way of example, ultraviolet lightis not expected to penetrate significantly beyond a tissue depth ofseveral microns for most tissue types, while visible light can beexpected to penetrate tens or thousands of microns, depending on thetissue type, and infrared light can be expected to penetrate millimetersor centimeters into various tissue types. The methods described hereincan be used to detect tissues and their components that occur beneath atsuch a distance from the optical probe that the tissue can be irradiatedand the relevant optical characteristic determined. At body locationscharacterized by the presence of multiple tissue types, noise andinterference can increase with distance of the assessed tissue from theprobe. For such locations, it is preferable to assess tissues located incontact with or very nearly opposed to the probe using radiation havinga penetrating capacity as low as necessary for effective assessment.

By way of example, a device described herein can be applied to a skinsurface (or to the oral surface of a human cheek or lip). The surfacecan be irradiated with near infrared (NIR) radiation. NIR reflected frommultiple regions of the surface can be assessed, and such assessmentwill reveal that some of the regions are relatively blood-rich and thatother regions exhibit reflectance characteristics more nearlycharacteristic of muscle. By way of example, differential NIRreflectance characteristics of hemoglobin and myoglobin can be used tomake this assessment as described by Schenkman et al., 1999, Appl.Spectrosc. 53(3):325-331. Once blood-rich and muscle-rich regions of thesurface have been identified, the blood rich regions may be furtheranalyzed for components of blood.

NIR Spectroscopy

NIR spectroscopy is a mature, non-contact, non-destructive analyticalcharacterization tool that has wide applicability to a broad range ofcompounds. The NIR region of the electromagnetic spectrum encompassesradiation with wavelengths of about 0.78 to 2.5 micrometers (i.e.,radiation with wavenumbers of 12,800 to 4,000 inverse centimeters, i.e.,12,800 to 4,000 cm⁻¹). NIR spectra result from the overtone andcombination bands of fundamental mid-infrared (MIR) bands.

NIR-based spectroscopy can be used to rapidly obtain both qualitativeand quantitative compositional information about the molecularcomposition of a material such as blood. NIR microscopes orspectrometers can be used to obtain NIR absorption, emission,transmittance, reflectance, or elastic scattering data at a singlewavelength or over a spectrum of wavelengths. NIR absorption data (e.g.,a spectrum) can be collected in transmittance, scattering, orreflectance mode.

NIR detectors have been used by others prior to this disclosure. Byusing optical filters (e.g., cold filters) to block visible wavelengths(ca. 0.4 to 0.78 micrometers), charge-coupled devices (CCDs, such asthose used in digital cameras and camcorders) can be used to detect NIRlight to wavelengths around 1100 nanometers. Other regions of the NIRspectrum can be viewed using devices such as indium gallium arsenide(InGaAs; ca. 0.9 to 1.7 micrometers) and indium antimonide (InSb; ca.1.0 to 5.0 micrometers) focal plane array (FPA) detectors. Integratedwavelength NIR imaging allow study of relative light intensities ofmaterials over broad ranges of the NIR spectrum. However, useful NIRspectral information can be unattainable without some type of discretewavelength filtering device.

The use of dielectric interference filters in combination with NIR FPAsis one method in which NIR spectral information can be obtained from anassessed region of a sample surface. To generate NIR spectralinformation, a NIR light beam is defocused to illuminate multipleregions of the sample surface (i.e., either individually, or by broadillumination of the surface) and the reflected, transmitted, orelastically scattered light from the illuminated area is transmitted toan NIR detector. A selection of discrete dielectric interference filters(provided in a filter wheel or in a linearly- or circularly-variableformat) can be positioned in front of a broadband NIR light source, orin front of the NIR FPA (i.e., between the illuminated area and the FPA)in order to collect NIR wavelength-resolved spectral information.Typically, the use of several fixed bandpass filters is required toaccess the entire NIR spectrum. Key limitations of the dielectric filterapproach include the need for a multitude of discrete filters to provideappreciable free spectral range, and the reliance on moving mechanicalparts in continuously tunable dielectric interference filters as arequirement to assess wavelength-resolved features. Although movingmechanical assemblies can be engineered, they add significant cost andcomplexity to NIR spectral analysis systems. Alternatives to movingmechanical assemblies can be more cost effective and provide performanceadvantages.

Acousto-optic tunable filters (AOTFs) have been employed in NIRspectrometers with substantially no moving parts. The AOTF is asolid-state device that is capable of filtering wavelengths from the UVto the mid-IR bands, depending on the choice of the filter's crystalmaterial. Operation of an AOTF is based on interaction of light with atraveling acoustic sound wave in an anisotropic crystal medium. Incidentlight is diffracted with a narrow spectral bandpass when a radiofrequency signal is applied to the device. By changing the applied radiofrequency (which can be under computer control, for example), thespectral passband can be tuned rapidly and without moving parts. Themethods and devices described herein are not limited to those using anAOTF. Numerous other optical filtering technologies (e.g., liquidcrystal tunable filters, photonic crystals, spectral diversity filters,and fiber array spectral translators) are available and can be employedas desired by a skilled artisan in this field.

Raman Spectroscopy

Raman spectroscopy provides information about the vibrational state ofmolecules. Many molecules have atomic bonds capable of existing in anumber of vibrational states. Such a molecule is able to absorb incidentradiation that matches a transition between two of its allowedvibrational states and to subsequently emit the radiation. Thesevibrational transitions exhibit characteristic energies that permitdefinition and characterization of the bonds that are present in acompound. Analysis of vibrational transitions therefore permitsspectroscopic molecular identification.

Most often, absorbed radiation is re-radiated at the same wavelength, aprocess designated Rayleigh or elastic scattering. In some instances,the re-radiated radiation can contain slightly more or slightly lessenergy than the absorbed radiation (depending on the allowablevibrational states and the initial and final vibrational states of themolecule). The energy difference is consumed by a transition betweenallowable vibrational states, and these vibrational transitions exhibitcharacteristic values for particular chemical bonds, which accounts forthe specificity of vibrational spectroscopies such as Ramanspectroscopy.

The result of the energy difference between the incident and re-radiatedradiation is manifested as a shift in the wavelength between theincident and re-radiated radiation, and the degree of difference isdesignated the Raman shift (RS), measured in units of wavenumber(inverse length). If the incident light is substantially monochromatic(single wavelength) as it is when using a laser source, the scatteredlight which differs in frequency can be more easily distinguished fromthe Rayleigh scattered light.

Because Raman spectroscopy is based on irradiation of a sample anddetection of scattered radiation, it can be employed non-invasively andnon-destructively, such that it is suitable for analysis of biologicalsamples in situ. Water exhibits relatively little Raman scattering(e.g., water exhibits significantly less Raman scattering than infraredabsorbance), and Raman spectroscopy techniques can be readily performedin aqueous environments. Raman spectral analysis can be used to assessoccurrence of and to quantify blood components and components of othertissues.

The Raman spectrum of a material can reveal the molecular composition ofthe material, including the specific functional groups present inorganic and inorganic molecules. Raman spectroscopy is useful fordetection of metabolites, pathogens, and pharmaceutical and otherchemical agents because most, if not all, of these agents exhibitcharacteristic ‘fingerprint’ Raman spectra, subject to various selectionrules, by which the agent can be identified. Raman peak position, peakshape, and adherence to selection rules can be used to determinemolecular (or cell) identity.

In the past several years, a number of key technologies have beenintroduced into wide use that have enabled scientists to largelyovercome the problems inherent to Raman spectroscopy. These technologiesinclude high efficiency solid-state lasers, efficient laser rejectionfilters, and silicon CCD detectors. In general, the wavelength andbandwidth of light used to illuminate the sample is not critical, solong as the other optical elements of the system operate in the samespectral range as the light source.

In order to detect Raman scattered light and to accurately determine theRaman shift of that light, the sample should be irradiated withsubstantially monochromatic light, such as light having a bandwidth notgreater than about 1.3 nanometers, and preferably not greater than 1.0,0.50, or 0.25 nanometer. Suitable sources include various lasers andpolychromatic light source-monochromator combinations. It is recognizedthat the bandwidth of the irradiating light, the resolution of thewavelength resolving element(s), and the spectral range of the detectordetermine how well a spectral feature can be observed, detected, ordistinguished from other spectral features. The combined properties ofthese elements (i.e., the light source, the filter, grating, or othermechanism used to distinguish Raman scattered light by wavelength)define the spectral resolution of the Raman signal detection system. Theknown relationships of these elements enable the skilled artisan toselect appropriate components in readily calculable ways. Limitations inspectral resolution of the system (e.g., limitations relating to thebandwidth of irradiating light, grating groove density, slit width,interferometer stepping, and other factors) can limit the ability toresolve, detect, or distinguish spectral features. The skilled artisanunderstands that and how the separation and shape of Raman scatteringsignals can determine the acceptable limits of spectral resolution forthe system for any of the Raman spectral features described herein.

Typically, a Raman peak that both is distinctive of the substance ofinterest and exhibits an acceptable signal-to-noise ratio will beselected. Multiple Raman shift values characteristic of the substancecan be assessed, as can the shape of a Raman spectral region that mayinclude multiple Raman peaks. If the sample includes unknown components,then the entire Raman spectrum can be scanned during spectral dataacquisition, so that the contributions of any contaminants to the datacan be assessed.

Devices

The invention includes devices for assessing a component of a tissue ofan animal by the methods described herein. The device comprises a firstdetector for detecting an optical characteristic of each of multipleirradiated regions of the body of the animal. The multiple regions canhave a pre-determined geometric relationship, which need not be aregular pattern nor even invariant from surface to surface. It issufficient that the regions retain their geometric relationship onlylong enough to permit correlation of optical properties of the tissue ofinterest and the tissue component for the regions. The device includes acontroller that is operably linked to the detector. The controllerrestricts detection of the optical property(ies) of the tissue componentto the regions that exhibit an optical characteristic of the tissue ofinterest.

The detectors used to assess an optical characteristic of a tissue andone or more optical properties of the tissue component can be, andpreferably are, a single detector. Numerous suitable detectors are knownin the art (e.g., CCD detectors), and selection of an appropriatedetector is within the ken of the ordinarily skilled artisan in view ofthe disclosure herein.

The device can include a radiation source for irradiating the regions ofthe sample, either individually (e.g., using optical fibers to transmitlight from the radiation source to the regions) or collectively (e.g.,by irradiating two or more regions with radiation transmitted from thesource or from an optical fiber optically coupled to the source).Multiple radiation sources can be included in (or packaged with) thedevice, each of the multiple sources irradiating some or all of theregions of the sample surface. Radiation sources which emit radiation ofdifferent wavelengths, for example, can be used where analyticaltechniques requiring such illumination is desired. Monochromatic orpolychromatic lights sources can be used. Selection of an appropriateradiation source can be made by an ordinarily skilled artisan in view ofthe other components of the device, the spectral techniques employed,and the disclosure herein.

Light reflected, transmitted, emitted, or scattered (elastically orinelastically) from the sample surface is delivered to one or moredetectors so that each region can be assessed for occurrence of anoptical property characteristic of the tissue of interest and so thatone or more regions that exhibit such a characteristic can be furtherassessed for occurrence, magnitude, or both, of one or more opticalproperties characteristic of the tissue component of interest. Thislight can be transmitted directly from the surface to the detector(e.g., using a detector that contacts the sample surface or has a layerof air or another substance interposed between it and the samplesurface). The light can be transmitted from the surface to the detectorusing optical fibers along some or all of the gap between the surfaceand the detector.

One or more optical elements (other than, or in addition to, opticaltransmission fibers) can be optically coupled with the detector andinterposed between the surface and the detector. Examples of appropriateelements include a lens, and an optical filter. For example, if thedevice employs Raman spectral analysis to assess occurrence of a tissuecomponent, it can be advisable to filter light scattered from the samplesurface to reduce (or preferably substantially eliminate) elasticallyscattered light from the radiation transmitted from the surface to thedetector.

The device can include a computer memory unit for storing information(e.g., reference spectra) useful for correlating Raman-shifted radiationscattered from the regions with concentration of the blood component.The memory unit can also store relevant optical property informationuseful for comparison with data gathered from the sample for the purposeof identifying portions of the animal body at or near which the tissueof interest occurs. The device can also include a display (e.g., anumerical display) for indicating an optical characteristic of thesample or the concentration of a tissue component, for example. A powersupply (e.g., a battery) can be incorporated into the device, or thedevice can be adapted for connection to an external power supply (e.g.,it can have a plug suitable for insertion into a standard residential orcommercial electrical wall socket).

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teaching providedherein.

Example 1

FIG. 1 is a schematic diagram of an embodiment of the non-invasivechemometric analysis device described herein. In FIG. 1, a probe 20 hasa surface 22 that can be applied to a vascularized surface of an animal.Multiple holes 24 extend through the surface, through which radiationcan pass (e.g., through air, a lens, or one or more optical fibers inthe hole). In the embodiment shown in FIG. 1, optical fibers forillumination 40 are optically coupled with a radiation source 60 andpass through the holes 24 in the probe 20 to illuminate whatever liesadjacent the surface 22. Optical fibers 50 for collecting lightreflected, emitted, or scattered from whatever lies adjacent the surface22 are optically coupled to a detector 80, optionally by way of anoptical element 70 such as a tunable filter or an interferometer. Thedetector 80 assesses light transmitted thereto by the optical fibers 50to determine an optical property corresponding to discrete regions ofspace adjacent the surface 22 (e.g., each region corresponding to asingle optical fiber or to a group of optical fibers). A computerprocessor or other controller 90 identifies regions for which theoptical property is characteristic of blood, optionally storing them ina computer memory unit 110. Using light transmitted from the identifiedregions by way of the optical fibers 50, the detector 80 can assess asecond optical property. In this manner, assessment of the secondoptical property can be limited to regions of space adjacent the surface22 that exhibit an optical characteristic of blood. The second opticalproperty can be used, for example, to assess the concentration of thecomponent in the blood, and that concentration can be calculated by thecomputer processor 90 and stored in memory 110, displayed on a display100, or both.

By way of example, the device illustrated in FIG. 1 can be used toassess blood glucose concentration in a human as follows. The surface 22of the probe 20 is placed against a vascularized surface (e.g., skin orunder the tongue) of the human. Radiation generated by the radiationsource 60 passes through the illumination delivery fibers 40, wherebythe vascularized surface is irradiated through multiples holes 24 in thesurface 22 into or through which the fibers 40 pass. Light reflectedfrom the human passes into optical fibers 50 that are present in orbehind, or extend through, the holes 24. In this embodiment, a liquidcrystal tunable filter 70 is tuned to pass light having a wavelength forwhich the reflectance characteristics of blood are known. The lightpasses to a CCD detector 80 at which the intensity of reflected light isassessed for multiple regions of the human. A computer 90 compares theintensity values with reference values stored in an operably connectedcomputer memory unit 110. For each region for which the intensity valueindicates the presence of a suitable amount of blood, the computer 90causes the detector 80 to assess one or more optical properties ofglucose, such as a Raman spectrum or the intensity of Raman-shiftedradiation scattered from the region at Raman shift values characteristicof glucose. The second optical property(ies) can be stored in the memory110 or used by the computer 90 to calculate a concentration of glucosein the blood.

In FIG. 1, the probe 20 is shown having holes 24 situated essentiallyrandomly across the surface 22 thereof. Although the holes 24 can bearranged essentially randomly, they can also be arranged in a regular orirregular pattern. Each of FIGS. 2 and 3 illustrates an alternativelayout of holes 24 across a cutaway portion of the surface 22 of theprobe 20. In FIG. 2, five holes 24 of a regular pattern are shown, eachhole 24 having nineteen optical fibers extending therethrough, includingone centrally-situated optical fiber 40 for transmitting light onto thesample surface and eighteen optical fibers 50 circumferentially arrangedaround optical fiber 40 for collecting and transmitting light from thesample surface. In FIG. 3, six holes 24 of a regular pattern are shown,each hole 24 having twenty-three optical fibers extending therethrough,including three centrally-situated optical fibers 40 for transmittinglight onto the sample surface and twenty optical fibers 50circumferentially arranged around optical fibers 40 for collecting andtransmitting light from the sample surface. However, the holes need notbe round, nor need the fibers 40 and 50 be arranged in a regularpattern. For example, FIG. 4 illustrates a hole 24 in the surface 22 ofthe probe 20 in which fourteen illuminating fibers 40 and thirty-ninelight-collecting fibers 50 are arranged in an essentially random array.

FIG. 5 illustrates two embodiments of how sample illuminating fibers 40and light-collecting fibers 50 can be situated within holes 24 thatextend through the surface 22 of the probe 20. In FIG. 5A, the ends ofthe illuminating fiber 40 and light-collecting fibers 50 aresubstantially flush with the surface 22. In FIG. 5B, the ends of fibers40 and 50 are optically coupled with a lens 30.

Example 2

Blood Glucose Determination

The invention described herein provides an integrated method to performa non-invasive, rapid measurement of the body chemistry of a consciousor unconscious person. The method involves using a small multipointprobe inserted under the tongue or into another vascularizedtissue-lined body surface or cavity. The probe can perform severalmeasurements simultaneously (e.g., under computer control) at severalpoints without a need to move the probe. Several features of the probe,where and how it is located and the laser excitation wavelength used,can be routinely optimized for Raman scattering assessment of glucoseand other blood components. Known spectral unmixing and other softwarealgorithms can be applied to the acquired Raman data and enhance theselectivity and sensitivity of the methods for detection andquantification of analytes. Such enhancements can improve the accuracyand reduce the time between sampling and production of an analysisreport.

Diabetes is recognized as a widespread health problem. Blood glucosemonitoring is critical for the treatment and medication of diabetes.Currently, glucose is usually monitored by breaking the skin to obtain ablood sample which is analyzed to determine relative concentrations ofspecific chemicals in the blood. Optical methods such as Raman and IRhave the capability to quantitatively characterize the chemicals in theblood, but suffer when applied non-invasively (without penetration ofthe skin) due to a number of complicating factors. For example, pasttrans-epidermal approaches suffer from the need to use long wavelengthsto penetrate the skin to detect chemicals or gases in the blood. Use ofshorter wavelengths makes detection of optical signals more complex andcostly. For Raman spectroscopy, the scattering cross sections at longerNIR wavelengths are significantly reduced relative to visiblewavelengths, typically by a factor of 10. Long collection times for alarge localized probe on a non-anesthetized patient is problematical,since normal movement of the patient will produce variations in signalintensities over time, and those variations distort spectral from thetarget area. In addition, variations in skin pigmentation can limit theinterpretation of the chemical information obtained from many opticalmethods. Such pigmentation variations require specific calibration ofdetected components for each individual measured.

Much prior art is directed to using optical measurements to measure theanalytes in blood or the composition of tissues removed from the bodyfor the purpose of pathology. Use of NIR and infrared (IR) spectroscopyto detect blood gases and blood analytes non-invasively has been longpursued, without any evidence of commercially viable products.

The methods and devices described herein provide an integrated approachthat optimizes Raman sample measurement and data analysis to minimizedata measurement times and patient discomfort. Efficient Ramanscattering analysis for specific target blood chemistry analytes can beobtained. The methods and devices permit a relatively non-skilledoperator, such as a physician, a physician's assistant, a nurse, or apatient, to perform the data acquisition. In one embodiment, severalpoints on a vascularized tissue surface are simultaneously measuredusing a series of high numerical aperture micro lens (i.e., ordinarylens or a fresnel lens) that are coupled to optical fibers for thedelivery of excitation radiation and collection of Raman scatteredlight. The multipoint micro-lens sampling head and fiber optic opticaldelivery system comprise the detection probe which can be attached to ahandheld detector or connected via a handheld extension to a portablesource/detection/analysis station.

To avoid complications found from the optical properties and variabilityof the compounds in the skin, such as melanin, a compact,thermometer-like multipoint probe can be inserted into a “pink tissue”body cavity. The tissues lining the cavity under the tongue have arevery thin epithelia, and multipoint sampling of these tissues canenhance illumination of blood rich tissues, such as blood vessels.Tissue lining the rectum is also rich in blood vessels and has arelatively thin epithelial lining. The geometry and orientation of themouth and extremities of the tongue enable the probe a to be fixedlyheld in place for the duration of a measurement, and clamps,mouthpieces, or similar adapters can be used to enhance fixedness ifnecessary. The under-tongue region also has the advantage that it is aconvenient and familiar location for the patient to hold a probe.

Example 3

Assessment of a Tissue Component in One or More Tissues

The devices described herein assess optical properties at multiple bodylocations. A plurality of tissue types can therefore be assessedsimultaneously using the devices, the number of assessable tissuesdepending on the number of tissue types that are contacted by or inclose opposition to a radiation-collecting optical conduit of thedevice. Careful placement of an optical probe described herein by askilled operator can increase or decrease the likelihood that aparticular tissue type will be contacted by the probe. Furthermore, ifdesired, probes can be constructed to maximize the likelihood that theprobe will contact a tissue of interest when the probe is applied to orinserted into the body of an animal.

FIG. 6 illustrates assessment of one or more tissue components inmultiple tissue types. The device used in the analysis illustrated inFIG. 6 includes an elongate probe 20 that, in FIG. 6, is emplaced in thebody of an animal. The probe 20 has a plurality of lenses 30 along itslength. Those lenses 30 contact, or are in close opposition to a varietyof tissue types. Not shown in FIG. 6 are optical fibers extending alongthe length of the probe 20 which transmit radiation collected by lenses30 to a detector.

In FIG. 6, lens 30-1 collects radiation from skin tissue S. Lens 30-2collects radiation from sub-dermal connective tissue C. At least twolenses, 30-3 and 30-4, collect radiation from muscle tissue M. Lenses30-5 and 30-6 collect radiation from venous V and arterial A capillarieswhich are located in close proximity to the lenses. Lens 30-7 collectsradiation from several tissues, including muscle M and connective Ctissues and from a fatty deposit F. To detect glucose in muscle cells,the device collects radiation from each of the lenses and assesses, foreach lens, whether the collected radiation exhibits a first opticalcharacteristic of muscle tissue. From lenses corresponding to muscletissue (i.e., lenses 30-3, 30-4, and 30-7), the device also collectsradiation and assesses that radiation for a second opticalcharacteristic of glucose. Glucose in muscle tissue can be quantified bycorrelating the second optical characteristic of radiation collectedfrom the lenses corresponding to muscle tissue using the known opticalproperties of glucose.

The preceding analysis included assessment of the second opticalcharacteristic for radiation collected by lens 30-7, which collectsradiation from muscle and two other tissues. An assessment of glucosemore specific for muscle tissue can be obtained by excluding from theanalysis radiation collected from body locations that exhibit both thefirst optical characteristic of muscle tissue and an opticalcharacteristic of another tissue type. In this more specific assessment,only radiation collected from lenses 30-3 and 30-4 is assessed for thesecond optical characteristic of glucose.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims include all such embodiments and equivalent variations.

1. A method of assessing a component of a tissue of an animal, themethod comprising: irradiating a plurality of regions of the tissue togenerate radiation therefrom; evaluating first optical data generatedfrom each of said plurality of irradiated regions to identify at leastone of said plurality of regions of tissue exhibiting a first opticalcharacteristic of the tissue, said first optical characteristicassociated with said first optical property; obtaining second opticaldata generated from said at least one identified region in saidplurality of regions, wherein said first and said second optical dataare generated by different spectroscopic modes, wherein the secondoptical data is generated by transmitting radiation, generated from saidat least one identified region, through a filter, thereby obtainingfiltered photons; assessing said component in said at least oneidentified region by evaluating said filtered photons for a secondoptical characteristic associated with said component; and reporting anidentity for said component in accordance with a result of saidaccessing step.
 2. The method of claim 1, wherein the component isassessed in the tissue in vivo.
 3. The method of claim 1, wherein thesecond optical characteristic of the component is assessed for at leasttwo identified regions.
 4. The method of claim 1, wherein the secondoptical characteristic is a Raman shift of light scattered from theidentified region that is attributable to the component.
 5. The methodof claim 1, wherein the first optical characteristics of the irradiatedregions are assessed using multiple optical conduits that opticallycouple the irradiated regions with a detector.
 6. The method of claim 5,wherein the conduits are optical fibers.
 7. The method of claim 6,wherein the optical fibers are arranged in a coherent bundle.
 8. Themethod of claim 5, wherein, for each irradiated region, the first andsecond optical characteristics are assessed using the same opticalconduit.
 9. The method of claim 5, wherein, for each irradiated region,the first and second optical characteristics are assessed using multipleoptical conduits.
 10. The method of claim 5, wherein the opticalconduits extend along a probe inserted into the body of the animal. 11.The method of claim 5, wherein the optical conduits extend along a probeapplied against a body surface of the animal.
 12. The method of claim 5,wherein the optical conduits are rigidly fixed relative to one another.13. The method of claim 1, wherein the component is a cell.
 14. Themethod of claim 13, wherein the component is an abnormal cell of thetissue.
 15. The method of claim 1, wherein the tissue comprises cells ofmultiple types.
 16. The method of claim 1, wherein the regions areirradiated with substantially monochromatic light.
 17. The method ofclaim 1, further comprising: applying a spectral unmixing method to saidsecond optical property to distinguish said second opticalcharacteristic associated with said component from other opticalcharacteristics associated with other components.
 18. A device forassessing a component of a tissue of an animal in vivo, the devicecomprising a radiation source for irradiating multiple regions of ananimal body, light being conveyed from the radiation source to each ofthe regions by at least one discrete optical illumination fiber for eachregion; a first detector for identifying regions that exhibit a firstoptical characteristic of the tissue, the first detector being opticallycoupled with the regions by way of at least one discrete opticaldetection fiber for each region; a second detector for assessingRaman-shifted radiation scattered by the identified regions, the seconddetector being optically coupled with the identified regions by way ofat least one discrete optical detection fiber for each region, and therebeing an optical filter interposed between the second detector and theidentified regions and; a computer processor operably linked to thesecond detector for restricting assessment of Raman-shifted scatteredradiation to the identified regions and for correlating Raman-shiftedradiation scattered by the identified regions with the quantity of thecomponent in the tissue.